1. Field of the Invention
The present invention relates to a process for purifying a human interferon beta from a recombinant human interferon beta-containing culture using affinity chromatography and reversed-phase high-performance liquid chromatography.
2. Description of the Related Art
Interferons in a broad meaning are extracellular messengers mediating reactivity of hosts and evolutionally conserved protein families that are released in a relatively small size from cells. Interferons are released from interferon-producing cells in response to stimulation by viruses, double-stranded RNAs, various microorganisms, or cytokines such as TNF or IL1, and then bind to surfaces of neighboring cells with interferon receptors. Thereafter, interferons induce synthesis of various proteins so that reactivity and homeostasis of hosts are maintained by consecutive signaling in the cells. Therefore, interferons act as antiviral, antiproliferative, and immune signaling proteins in the bodies and have direct antiproliferation effects on cancer cells, and thus, have received much attention as therapeutic agents [Postka S., Langer J. A. and Zoon K. C. (1987) Interferons and their actions, Annu. Rev. Biochem. 56:727-777].
Interferons belong to the class of helical, physiologically active substances. According to physicochemical characteristics and functionalities, there are two classes of interferons: type 1 and 2. Interferon-alpha, -beta, -tau, and -epsilon are members of the type 1 interferon [Weissman C. and Weber H. (1986) The Interferon genes, Prog. Nucleic Acid Res. Mol. Biol. 33:251-300] and interferon gamma is a member of the type 2 interferon. Among them, interferon betas belonging to the type 1 interferon are proteins that exhibit species specificity. Interferon betas are also called as fibroblast interferons considering their sources and as pH2-stable interferons considering biological characteristics. Interferon betas bind to the same receptors of cell surfaces, together with interferon alphas belonging to the type 1 interferon, and then induce transcription of antiviral factors in response to a consecutive cell signaling system.
Interferon betas are glycoproteins (about 20% sugar moiety) with a molecular mass of about 20 kDa and single-chain proteins consisting of 166 amino acids. One N-glycosylation site is known to play a role in increasing material stability or solubility as physicochemical functions, rather than participating in biological activity or antigenicity [Karpusas M., Whytty A., Runkel L., and Hochman P. The structure of human interferon-β: implications for activity CMLS, 54:1203-1216 1998].
Advance in genetic recombination technology enabled determination of the amino acid sequence of human interferon beta and cloning and expression of human interferon beta in E. coli [Taniguchi, Gene 10:11-15, 1980]. Furthermore, expression of interferon beta in Chinese hamster ovary (CHO) cells was also reported [U.S. Pat. No. 4,966,843, U.S. Pat. No. 5,376,567, and U.S. Pat. No. 5,795,779].
Currently, interferon betas are manufactured by gene recombination technology and commercially available under the trade name of Betaseron®, Avonex®, and Rebif®. Recombinant interferon betas are known to be effective in delaying the progression of multiple sclerosis in patients with the signs of the disease and relieving the pains of the disease. Furthermore, recombinant interferon betas are widely used as therapeutic agents for multiple sclerosis, and at the same time are effective in nonspecific regulation of human immune response, immune response to viral infection, and anti-proliferation of cancer cells.
Currently available purification technologies of recombinant interferon betas expressed in CHO cells involve 3-5 purification procedures including primary purification by affinity chromatography (U.S. Pat. No. 4,278,661, U.S. Pat. No. 4,289,689, U.S. Pat. No. 4,541,952, U.S. Pat. No. 4,808,523, etc.), metal-chelate chromatography (U.S. Pat. No. 4,257,938, U.S. Pat. No. 4,359,389, U.S. Pat. No. 4,541,952, U.S. Pat. No. 5,244,655, etc.), CPG (controlled pore glass) chromatography (U.S. Pat. No. 4,359,389, U.S. Pat. No. 5,066,786, U.S. Pat. No. 5,244,655, etc.), or Concanavalin A chromatography (U.S. Pat. No. 4,289,689, U.S. Pat. No. 4,658,017, etc.) followed by cation exchange chromatography and reversed-phase chromatography.
In the above-described common purification technologies, metal-chelate chromatography may cause environmental contamination due to use of heavy metal. CPG or Concanavalin A chromatography has poor purification specificity. That is, Concanavalin A chromatography based on selective binding with many sugar-chain proteins contained in a CHO cell culture exhibits low specificity. A CPG column allows separation by molecular size after binding with a protein. However, separation efficiency and purity of interferon betas are lower than those by affinity chromatography (e.g., Blue Sepharose column chromatography).
Furthermore, common purification technologies by affinity chromatography involve washing and elution with ethylene glycol using a monoclonal antibody and/or a dye-resin. However, affinity chromatography using a monoclonal antibody separately requires the removal of the nonglycosylated form of interferon beta, which renders mass production difficult. In particular, ethylene glycol used in washing and elution is very toxic in the body, which restricts actual purification application.
Meanwhile, U.S. Pat. No. 4,483,849 discloses a process for purifying and stabilizing interferon beta using propylene glycol, instead of toxic ethylene glycol, by affinity chromatography. The process disclosed in this patent document includes applying an interferon-containing culture to a dye-affinity column such as equilibrated Affi-Gel Blue, washing the column with a 1.0M NaCl/PO4 buffer solution and a 1.0M NaCl/PO4 buffer solution containing 40% propylene glycol, and eluting interferon with 50% propylene glycol. Even though the process of this patent document involves column washing and elution, a desired final product peak and an impurity peak coexist, thereby lowering purity.